Keywords: amphotericin B resistant and susceptible A. terreus strain cluster, Galleria mellonella, invertebrate in vivo modelThe aim of this study was to investigate if the alternative in vivo model Galleria mellonella can be used (i) to determine differences in pathogenicity of amphotericin B (AMB) resistant and susceptible A. terreus isolates, (ii) to evaluate AMB efficacy in vivo (iii) and to correlate outcome to in vitro susceptibility data. Larvae were infected with 2 A. terreus AMB resistant (ATR) and 3 AMB susceptible (ATS) isolates and survival rates were correlated to physiological attributes and killing ability of larval haemocytes. Additionally, infected larvae were treated with different concentrations of L-AMB. Haemocyte density were ascertained to evaluate the influence of L-AMB on the larval immune cells. Larvae were sensitive to A. terreus infection in an inoculum-size and temperature dependent manner. In vitro susceptibility to L-AMB correlated with in vivo outcome of antifungal treatment, defining an AMB susceptible strain cluster of A. terreus. Susceptibility to L-AMB increased virulence potential in the larval model, but this increase was also in accordance with faster growth and less damage caused by larval haemocytes. L-AMB treatment primed the larval immune response by increasing haemocyte density. G. mellonella provides a convenient model for the in vivo screening of A. terreus virulence and treatment options, contributing to the generation of a hypothesis that can be further tested in refined experiments in mammalian models.

Galleria mellonella larvae are widely used for assessing the virulence of microbial pathogens and for measuring the in vivo activity of antimicrobial agents and produce results comparable to those that can be obtained using mammals. The aim of the work described here was to ascertain the effect of pre-incubation at 15°C for 1, 3, 6 or 10 weeks on the susceptibility of larvae to infection with Candida albicans and Staphylococcus aureus. Larvae infected with C. albicans after 1 week pre-incubation at 15°C showed 73.3 ± 3.3% survival at 24 hours post-infection while those infected after 10 weeks pre-incubation showed 30 ± 3.3% survival (P < 0.01). Larvae infected with S. aureus after 1 week pre-incubation showed 65.5 ± 3.3% survival after 24 hours while those infected after 10 weeks pre-incubation showed 13.3 ± 3.3% (P < 0.001). Analysis of the haemocyte density in larvae pre-incubated for 3-10 weeks showed a reduction in haemocytes over time but a proportionate increase in the density of granular haemocytes in the population as determined by FACS analysis. Proteomic analysis revealed decreased abundance of proteins associated with metabolic pathways (e.g. malate dehydrogenase, fructose-1,6-bisphosphatase, glyceraldehyde-3-phosphate dehydrogenase) and prophenoloxidase. G. mellonella larvae are a useful in vivo model system but the duration of the pre-incubation stage significantly affects their susceptibility to microbial pathogens possibly as a result of altered metabolism.

a b s t r a c tExposure of larvae of Galleria mellonella larvae to mild physical (i.e. shaking) or thermal stress for 24 h increased their ability to survive infection with Aspergillus fumigatus conidia however larvae stressed in a similar manner but incubated for 72 h prior to infection showed no elevation in their resistance to infection with A. fumigatus. Stressed larvae demonstrated an elevated haemocyte density 24 h after initiation of the stress event but this declined at 48 and 72 h. Larval proteins such as apolipophorin, arylophorin and prophenoloxidase demonstrated elevated expression at 24 h but not at 72 h. Larvae maintained at 37°C showed increased expression of a range of antimicrobial and immune-related proteins at 24 h but these decreased in expression thereafter. The results presented here indicate that G. mellonella larvae are capable of altering their immune response following exposure to mild thermal or physical stress to mount a response capable of counteracting microbial infection which reaches a peak 24 h after the initiation of the priming event and then declines by 72 h. A short-term immune priming effect may serve to prevent infection but maintaining an immune priming effect for longer periods may be metabolically costly and unnecessary while living within the colony of another insect.

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The haemophagous mite, Varroa destuctor is one of the most dangerous threats to the Western honey bee, Apis mellifera. Varroa mites parasitize the larval and adult stages of the honey bee and can have devastating effects on the health of the individual bee and colony. In recent years, varroa have shown resistance to the pyrethroid group of insecticides, including Bayvarol ® which has flumethrin as the active ingredient. In the work presented here, changes in the expressed proteomes of mites, either sensitive or resistant to Bayvarol ® were observed using 2D-SDS-PAGE and shotgun label-free proteomics. A number of detoxification proteins (e.g., glutathione-s-transferase, flavin-containing monooxygenase) were present at higher levels in the resistant mites, as were some proton pumping proteins (e.g., Na+/K+ ATPase alpha and beta subunit, E1-E2 ATPase protein). A decrease in the abundance of 12 cuticle proteins in the resistant mites was observed indicating that alteration to cuticle structure could be a potential resistance mechanism. A number of structural proteins such as myosin and alpha tubulin were expressed at higher levels in the resistant mites, which could indicate a change to the intracellular structure of the cuticle barrier or a change in the cell shape/surface, rather than the addition of extra cuticle proteins. The results presented here indicate higher levels of protein associated with cellular detoxification in Bayvarol ® -resistant varroa mites.Análisis proteó mico de los mecanismos de resistencia a Bayvarol ® en el parásito de la abeja de la miel Varroa destructor El ácaro hemó fago, Varroa destuctor es una de las amenazas más peligrosas para la abeja occidental de la miel, Apis mellifera. Los ácaros varroa parasitan los estadios larvarios y adultos de la abeja de la miel y pueden tener efectos devastadores sobre la salud de la abeja y la colonia individual. En los últimos años, Varroa han mostrado resistencia al grupo de insecticidas piretroides, incluyendo Bayvarol ® que tiene la flumetrina como ingrediente activo. En el trabajo que aquí se presenta, se observaron cambios en los proteomas expresados por los ácaros, ya sea sensibles o resistentes a Bayvarol ® utilizando 2D-SDS-PAGE y proteó mica con marcaje libre shotgun. Ciertas proteínas de desintoxicació n (por ejemplo, transferasa-s-glutatió n, monooxigenasa incluyendo flavina) estaban presentes en niveles más altos en los ácaros resistentes, así como algunas proteínas de bombeo de protones (por ejemplo, las subunidades alfa y beta de la ATPasa Na + / K +, y la proteína ATPasa E1-E2). Se observó una disminució n en la abundancia de 12 proteínas de la cutícula de los ácaros resistentes lo que indica que la alteració n de la estructura de la cutícula podría ser un mecanismo de resistencia potencial. Ciertas proteínas estructurales, tales como la miosina y la alfa tubulina se expresaron en niveles más altos en los ácaros resistentes, lo que podrían indicar un cambio en la estructura intracelular de la barrera de la cutícula o un cambio en la form...